A rapid high-throughput device to isolate antibiofilm bacteriophage

Multi-drug resistant (MDR) bacterial infections are a global public health crisis with ESKAPEE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp, and Escherichia coli.) pathogens particularly troublesome. Wound infection is usually acquired in the hospital setting and is a significant source of morbidity and mortality in military healthcare. Empiric antibiotic treatment although previously preferred is now problematic due to the global rise in MDR isolates. With few new antibiotic solutions, new treatment strategies are needed. Therapeutic phage is an ideal alternative treatment because of their inherent bacterial specificity. Chronic bacterial infections are often accompanied with biofilm formations and many MDR organisms grow in biofilms possibly defeating phages isolated with planktonic bacteria. For phage therapy to be effective, it is necessary to identify phages capable of productively infecting a given biofilm bacterium. Although, assays that detect biofilm-disrupting agents have been developed, this technology has not been translated to searching for phage with antibiofilm activity. This proposal, by Guild BioSciences and The Ohio State University, will address this need with the development of a novel rapid high-throughput bead biofilm (HTBB) phage screening system that selects phages capable of disrupting biofilms. It will also be simple, inexpensive, and rapid antibiofilm phage hunting device (AB-PHD); thus, it will be a major advancement compared to existing traditional phage hunting. The critical novelty is a species-independent signaling component (SISC) based on fluorescent beads (fBs) that will be incorporated into biofilms during their initiation. If test phage/environmental phage lysates degrade biofilms, the SISC will be released to the supernatant and the resulting fluorescent signal will be measured. A bead-based biofilm assay established by our research institution collaborator Dr. Daniel Wozniak, will be the starting point for assay design. This proposal will select and develop a fluorescent bead based system using a Pseudomonas aeruginosa model system through three Technical Objectives: Develop a high throughput bead biofilm (HTBB) assay for antibiofilm phage screening. The test tube assay will be converted into a 96-well plate assay and optimized for detecting antibiofilm phage. 1. Develop and optimize the species-independent signaling component (SISC). Two classes of fBs will be initially evaluated for incorporation into biofilms, incidental uptake by bacteria, and release due to antibiofilm activity of first enzymes and then model phages. 2. Assess the performance of the combined SISC-HTBB system. The HTBB assay from TO1 and the SISC from TO2 will be combined, optimized and tested using model phage and environmental phage lysates. The use of biofilm beads for propagating/amplifying phage will be tested as well.